Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma . The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF . BRAF mutations may be critical for the initiation of melanoma ; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce melanoma.

Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo.[Keywords: Stem cell; Notch; Runx; AGM; zebrafish; irradiation] Supplemental material is available at http://www.genesdev.org.

The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell ( IntroductionThe study of the biologic effects of ␥ irradiation began over a century ago, 1 but it was not until deployment of the atomic bomb in 1945 that the clinical manifestations of lethal total body irradiation (TBI) were fully realized. Acute irradiation injury was replicated using animal models, and seminal experiments by Jacobsen et al 2,3 and Lorenz et al 4 showed that shielding or transplantation of hematopoietic tissues was sufficient for radioprotection of otherwise lethal doses. Although humoral factors produced from these tissues were initially proposed to be the radioprotective elements, 2 subsequent studies showed that survival correlated with the level of hematopoietic repopulation by donor-derived cells. [5][6][7][8] Pioneering studies by Till and McCulloch and colleagues showed that donor bone marrow contained rare cells that generated clonal, macroscopic spleen colonies, 9,10 a fraction of which regenerated spleen colonies 11 or repopulated multilineage hematopoiesis 12 in hosts receiving serial transplants. These experiments led to the concept of the hematopoietic stem cell (HSC).TBI has since been used to condition hosts prior to transplantation of donor bone marrow, both therapeutically in humans and as a means of testing murine blood cell subsets for the presence of HSCs. The development of monoclonal antibodies and multiparameter flow cytometry, combined with hematopoietic cell transplantation (HCT), has led to the prospective isolation of murine HSCs, [13][14][15][16] committed lymphoid 17 and myeloerythroid 18 progenitors, and their downstream progeny by cell-surface phenotypes. These powerful techniques have allowed individual components of the immune system to be tested functionally, which has led to the dissection of the hematopoietic hierarchy and the resolution of effector cell function. HCT also provides the means to study the effects of genetic mutations in isolated cell types when introduced into a normal cellular environment.The zebrafish has recently emerged as a unique vertebrate model in which forward genetic screens can uncover novel genes involved in blood cell development and function. 19 Many zebrafish blood mutants identified in these screens are embryonic lethal, and we have recently described embryonic HCT to address issues of cell autonomous mutant gene function. 20 Similar transplantation of whole kidney marrow (WKM) cells into unconditioned adult recipients resulted in the disappearance of donor-derived cells within several weeks of transplantation. This suggests that donorderived cells are either rejected by the host immune system or that engraftment of donor cells requires the creation of niche spa...